Chlamydia spp. development is differentially altered by treatment with the LpxC inhibitor LPC-011.

BACKGROUND: Chlamydia species are obligate intracellular bacteria that infect a broad range of mammalian hosts. Members of related genera are pathogens of a variety of vertebrate and invertebrate species. Despite the diversity of Chlamydia, all species contain an outer membrane lipooligosaccharide (LOS) that is comprised of a genus-conserved, and genus-defining, trisaccharide 3-deoxy-D-manno-oct-2-ulosonic acid Kdo region. Recent studies with lipopolysaccharide inhibitors demonstrate that LOS is important for the C. trachomatis developmental cycle during RB- > EB differentiation. Here, we explore the effects of one of these inhibitors, LPC-011, on the developmental cycle of five chlamydial species. RESULTS: Sensitivity to the drug varied in some of the species and was conserved between others. We observed that inhibition of LOS biosynthesis in some chlamydial species induced formation of aberrant reticulate bodies, while in other species, no change was observed to the reticulate body. However, loss of LOS production prevented completion of the chlamydial reproductive cycle in all species tested. In previous studies we found that C. trachomatis and C. caviae infection enhances MHC class I antigen presentation of a model self-peptide. We find that treatment with LPC-011 prevents enhanced host-peptide presentation induced by infection with all chlamydial-species tested. CONCLUSIONS: The data demonstrate that LOS synthesis is necessary for production of infectious progeny and inhibition of LOS synthesis induces aberrancy in certain chlamydial species, which has important implications for the use of LOS synthesis inhibitors as potential antibiotics.

Effect of exogenous frequency exposure on human cells.

Effects of exogenous bioresonance oscillations on biological and morphological characteristics of continuous human lung and liver cells were studied. Proliferative activities and viability of cells decreased after exposure to frequencies of 8 and 78.5 Hz. This was paralleled by cytopathic disorders in lung and liver cells. The frequency of 72 Hz exhibited a less intense effect on both cell types. Cell contamination with mycoplasmas was provisionally suppressed after cell exposure according to F44 and F45 programs and to 97.5 and 69.5 Hz frequencies.

New pharmacokinetic in vitro model for studies of antibiotic activity against intracellular microorganisms.

The capacity for intracellular growth is an important survival strategy for a large group of common pathogens. Helicobacter pylori, the etiological agent for gastritis and duodenal ulcer, has been shown by both in vivo and in vitro studies to have the capacity to invade epithelial cells. In vitro models are used to study the effect of antibiotics on microoganisms. Most investigations are performed in broth culture or on agar plates, but kinetic models for bacteria in broth have been described. We present a new, kinetic model adapted for intracellular pathogens. A glass chamber, with a metal rack fitting Falcon cell culture inserts, was connected to a pump by rubber tubes. Different tube diameters and pump speeds were evaluated, and the assay was designed to mimic the half-lives of the antibiotics in vivo, i.e., 11.5 h for azithromycin, 5 h for clarithromycin, and 1 h for amoxicillin. Monolayers of HEp-2 cells were grown in the inserts for 2 days, after which H. pylori (clinical strain 88-23), was added to the system. Internalization was allowed for 12 h, and extracellular H. pylori cells were eradicated with gentamicin. The inserts were moved to the glass chamber, containing medium with 12.5 mg of either amoxicillin or azithromycin per liter or 2.4 mg of clarithromycin per liter. This represents 12.5, 50, and 80 times the extracellular minimum bactericidal concentration value, respectively. Samples were taken at 0, 2, 4, 6, 8, and 24 h. The HEp-2 cells were lysed, and intracellular bacteria were counted by plating. Inserts with infected cells grown in drug-free medium were included as controls for each time interval. A 3-log10 reduction of H. pylori was achieved in the experiments with azithromycin, and a 4-log10 reduction was achieved in the clarithromycin experiments, while no intracellular effect was seen when amoxicillin was used. The antibiotic concentrations at the sampling intervals were 12.5, 3.1, 0.8, 0.2, 0.05, and 0 mg/liter for amoxicillin; 12.5, 11.5, 10, 9, 8, and 3 mg/liter for azithromycin; and 2.4, 1.8, 1.4, 1, 0.8, and 0 mg/liter for clarithromycin. This new model for pharmacokinetic studies provides a useful tool, with applications for a broad range of microorganisms.

Structure and function of the polypeptides in simian virus 40. I. Existence of subviral deoxynucleoprotein complexes.

Velocity sedimentation analysis of simian virus 40 degraded in alkaline buffers, pH 10.5, yields two components: soluble protein containing the largest polypeptides, VP1 and VP2, of the virion, and a deoxynucleoprotein complex (DNP-I) containing the viral deoxyribonucleic acid (DNA) and the small polypeptides, VP4, 5, and 6, and all or part of VP3. Dissociation of DNP-I by equilibrium centrifugation in CsCl yields a complex (DNP-II) of the viral DNA and residual, tightly bound polypeptide; VP4, 5, and 6, but not VP3, are recovered after separation from DNP-II. Treatment of the virus with beta-mercaptoethanol and iodination experiments suggest that VP1 and VP2 might exist as compact structures cross-linked with disulfide bonds, perhaps forming the capsid. VP4, 5, and 6 form a relatively stable complex with the viral DNA and are supposed to be the internal proteins. The location of VP3 is not well defined; at least a portion of it is tightly bound to the viral DNA.